Human Amniotic Epithelial Cells as a Tool to Investigate the Effects of Cyanidin 3-O-Glucoside on Cell Differentiation

Cyanidin, a kind of anthocyanin, has been reported to have chemotherapeutic activities in humans. Human amniotic epithelial cells (hAECs) are considered a potential source of pluripotent stem cells. hAECs have been used as a novel tool in regenerative cellular therapy and cell differentiation studies. In this study, to explore the effects of cyanidin-3-O-glucoside (Cy3G) on hAECs and their mechanisms, we investigated the transcriptomic changes in the Cy3G-treated cells using microarray analysis. Among the differentially expressed genes (Fold change > 1.1; p-value < 0.05), 109 genes were upregulated and 232 were downregulated. Ratios of upregulated and downregulated genes were 0.22% and 0.47% of the total expressed genes, respectively. Next, we explored the enriched gene ontology, i.e., the biological process, molecular function, and cellular component of the 37 upregulated (>1.3-fold change) and 124 downregulated (<1.3-fold change) genes. Significantly enriched biological processes by the upregulated genes included “response to muscle activity,” and the genes involved in this gene ontology (GO) were Metrnl and SRD5A1, which function in the adipocyte. On the other hand, the cell cycle biological process was significantly enriched by the downregulated genes, including some from the SMC gene family. An adipogenesis-associated gene DDX6 was also included in the cell cycle biological process. Thus, our findings suggest the prospects of Cy3G in modulating adipocyte differentiation in hAECs.


Introduction
In recent years, demands in foods rich in phytochemicals have increased [1]. Based on considerable epidemiological evidence, the presence of polyphenols has been proven to promote health and diminish the risk of various diseases such as cancers and cardiovascular diseases, etc. [2,3]. Polyphenols are contained in various vegetables and fruits [4]. Diets rich in polyphenols lead to the reduction of risks of several diseases [5,6]. To investigate the unknown functions of several phytochemicals beneficial to human health, employment of high-throughput systems based on mammalian cells is one of the best ways.
Human amniotic epithelial cells (hAECs) are considered one of the novel sources of pluripotent stem cells (PSCs). In recent years, growing attention has been given to the use of perinatal stem cells such as hAECs in the field of regenerative cellular therapy and cell differentiation studies. hAECs have some advantages over other PSCs in that they are isolated from discarded term placenta, which is a medical waste product and have similar pluripotent and multipoint properties of stem cells [7]. Therefore, hAECs are suitable

Changes of Gene Expression in Cy3G-Treated hAECs
Five upregulated genes showed changes in the expression more than 1.45-fold (Table 1). To know the detailed features of their encoded proteins, we checked their function in the UniProt database (https://www.uniprot.org/, accessed on 16 April 2020). The highest expressed gene was fructosamine 3 kinase-related protein (FN3KRP), which is involved in protein deglycation by mediating phosphorylation of fructoselysine residues on glycated protein to generate fructoselysine-3 phosphate [25]. Another important upregulated gene was meteorin-like protein (METRNL), which is a hormone and is induced following exercise or cold exposure and promotes energy expenditure. It is also able to stimulate energy expenditure associated with the browning of white fat depots and thus improves glucose tolerance [26]. Ras-related protein Rab-6A (RAB6A) is a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum and has a low GTPase activity. From gene ontology, RAB6A was also predicted to bind to myosin V [27]. PSMB8 (20S proteasome subunit beta type-8) has an ATP/ubiquitin-dependent proteolytic activity and processes the class I MHC peptides as the immunoproteasome [28]. DCTN4 (dynactin subunit 4), which is a dynactin subunit p62, has been identified as an interacting partner of the P-type ATPase, ATP7B protein in mammals [29].
On the other hand, 19 downregulated genes showed fold change more than −2.0 ( Table 2). Among the downregulated genes, DDX6 (DEAD box helicase 6), plays a role in the process of mRNA degradation [30]. The encoded protein of ZC3H11A (Zinc finger CCCH domain-containing protein 11A) is involved in nuclear mRNA export [31]. PRDM2 (PRdomain zinc finger protein 2) encoded protein is an S-adenosyl -L-methionine-dependent histone methyltransferase that explicitly methylates Lys-9 or histone H3 and may function as a DNA-binding transcription factor [32].

Discussion
Cyanidin has several bioactivities in mammals. Previous reports indicate that cyanidin may affect the differentiation of PSCs and adult stem cells. Cy3G has been reported to regulate the differentiation of RANKL-induced osteoclasts, which are multi-nucleated cells derived from hematopoietic stem cells (HSCs), and enhance cell fusion [33]. LnCap and DU145 human prostate cancer cells treated with Cyanidin-3-O-β-glucopyranoside showed anti-proliferative and pro-differentiation properties [17]. However, the effects of cyanidin on hAECs have not been investigated.
Enrichment analysis of Cy3G-treated hAECs showed that response to muscle activityrelated genes, namely METRNL and SRD5A1, were upregulated (Table 3). Metrnl is reported to have multifaceted functions in adipocyte differentiation concerned to brown fat cell differentiation of adipocyte [34]. Brown adipose tissue can dissipate energy compared to white adipose tissue. White adipose tissue possesses the capacity to generate brown-like adipocytes, termed beiging [35]. Metrnl was identified as a peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α4-dependent myokine and can be induced in muscle after exercise. Increased circulating levels of Metrnl indicates stimulated energy expenditure and improved glucose tolerance [34]. These changes are activated by the adaptive responses of cells to the environmental or intracellular stress stimuli, thus counteracting the stress and promoting cell survival [36]. Overexpressed Metrnl also upregulated the peroxisome proliferator-activated receptor gamma (PPARγ) and anti-inflammatory cytokines in white adipocytes [34].
Interestingly, in the skeletal muscle of Cy3G-administered mice, PGC-1α was upregulated via cyclic AMP (cAMP) elevation, which subsequently enhanced exercise performance [19]. Cy3G also induces the differentiation of the beige phenotypes via elevations of cAMP level, PGC-1α, and PPARγ in mice adipocyte cells [19]. These previous reports support our findings in transcriptome data using Cy3G-treated hAECs.
Another gene, SRD5A1, converts testosterone into 5-alpha-dihydrotestosterone and progesterone or corticosterone into their corresponding 5-alpha-3-oxosteroids, and has a central role in sexual differentiation and androgen physiology. Male mouse that lacked 5alpha-reductase type 1 (SRD5A1) has reduced bone and forelimb muscle grip strength [37]. Expression of SRD5A1 was found in human adipose tissues, where it shows a tendency to increase slightly with differentiation in preadipocytes [38].
PSMB8, a catalytic subunit for immunoproteasomes, also regulates the differentiation of preadipocytes and the differentiation of preadipocytes to mature adipocytes in mice. Psmb8−/− mice show reduced weight gain caused by a reduction in adipose tissue volume and small size of mature adipocytes. In addition, inhibition of Psmb8 in 3T3L1 mice adipocyte cells could disrupt the differentiation to mature cells [39].
In our study, enrichment analysis also showed upregulation of genes related to regulation of cytoskeleton organization such as CAPN2 and STMN1 (Table 3). CAPN2 encodes calpain-2 catalytic subunit, which is a calcium-regulated non-lysosomal thiol-protease. It catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction [40]. STMN1 encodes Stathmin, which is involved in the regulation of the microtubule filament system by destabilizing microtubules. Cytoskeleton remodeling is one of the first steps for the morphological transition from preadipocyte to mature adipocyte. Acetylation of α-tubulin is related to adipogenesis in mice and 3T3-L1 cells [41].
A downregulated gene, DDX6, which encodes DEAD-box helicase 6 protein, has been reported to function in adipogenesis. In human adipose tissue-derived stem cells, the number of DDX6 granules per cell was reduced during adipogenesis [42].
Enrichment analysis showed cell cycle-related genes were downregulated in the Cy3G-treated hAECs. This gene set includes 21 genes as follows: YMHAE, EIF4G1, SMC1A, TENT4B, PRPF40A, SON, CNOT4, WASL, AKAP9, MEI1, GOGGA2, CEP57, PCM1, CDK12, SMC5, TLK1, WAC, CBX5, RMI1, SPIN1, and MGA. CDK12, which encodes cycle dependent kinase 12, is associated with elongating RNA polymerase II and its activity is required for G1/S progression [43]. SMC1A encodes a protein of central component of chromosome cohesion complex during cell cycles [44]. Cell cycle is an important event during cell proliferation but not necessary during cell differentiation. The length of G1 phase arrest is important for the decision of proliferation or differentiation [45]. In Cy3G-treated AECs, downregulation of genes related to G1/S progression may be indicated of the induction of differentiation in hAECs.
Previously, Kim et al. (2012) reported that anthocyanin extracts from black soybeans comprising Cy3G (68.3%) as well as delphinidin-3-O-glucoside (25.2%) and petunidin-3-O-glucoside (6.5%) could inhibit adipocyte differentiation and basal lipolysis in 3T3-L1 cell line [46]. Another study by Jeon et al. (2015) reported that the ethanol testa-extract of black soybean might suppress the differentiation of subcutaneous adipose-derived stem cells into the precursor cells of adipocytes [47]. However, adipocyte phenotype was studied in neither of the studies. We have reported in our previous studies that Cy3G, both synthetic and derived from black soybeans, could induce beige phenotype of adipocyte differentiation in mouse preadipocyte 3T3-L1 cells and improves insulin resistance [3,20].
Natural bioactive compounds have great potential to induce the targeted differentiation of stem cells in a lineage-specific manner by modulating cellular behavior and early biological and molecular events; however, only a handful of studies explored it. In our previous studies, we have reported that a caffeic acid ester (rosmarinic acid), a caffeoylquinic acid derivative (TCQA), and an iridoid glycoside (verbenalin) directed the differentiation of hAECs towards neuronal-lineage, whereas a flavonol aglycone isorhamnetin induced hepatic-lineage specific differentiation in hAECs [8][9][10][11]. We assume that other compounds from the anthocyanin group may have a similar effect as Cy3G on hAEC's fate choice. Their efficacy may vary depending on the presence or absence as well as the position of the sugar moieties. Therefore, the role of different anthocyanins on modulating adipocyte differentiation in hAECs should be explored further. Additionally, assessments of the effects of Cy3G on protein levels is required to confirm our observational transcriptomic analysis findings.
Altogether, our findings suggest that Cy3G may have prospects in inducing adipocyte differentiation in hAECs. Although hAECs have multilineage differentiation potential [7,48], their adipogenic differentiation is still controversial. While some reported adipogenic differentiation of hAECs under proper culture conditions [49][50][51], others did not find any adipogenic differentiation potential of hAECs [52,53]. Therefore, adipocyte differentiationenhancing effects of Cy3G, a plant-based compound, in hAECs in the absence of any growth factors and cytokines, may have an important step towards clinical applications of hAECs in reconstructive, corrective, and cosmetic fields. However, further in-depth investigation to validate our primary findings is warranted.

Extraction of AECs and Cell Culture Maintenance
Isolation of amnion epithelial cells (AECs) and their culturing was followed by the methods described previously [8,9]. In brief, the amnion was aseptically separated from the chorion and washed with Hank's Basic Salt Solution -Calcium and Magnesium Free (CMF-HBSS, Wako Pure Chemical Industries Ltd., Osaka, Japan). The smaller pieces of amnion were treated with a pre-digestion buffer (CMF-HBSS with EGTA, Wako Pure Chemical Industries Ltd., Osaka, Japan), rocked in the solution, and incubated for 10 min at 37 • C. After incubation, the Trypsin-EDTA was added to the tissue and incubated for 40 min at 37 • C and then transferred on ice. Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, St.-Louis, MI, USA) with FBS (Thermo Fisher Scientific Inc., Waltham, MA, USA) and penicillin-streptomycin (Lonza Walkersville Inc., Walkersville, MD, USA) was added to the trypsin digest. After centrifugation, pellets were resuspended and filtered through a 100 mm filter. The cell suspension was collected.
To maintain the AECs, the cells were cultured in Placenta Epithelial Cell Basal Medium (PromoCell, Cat. #C-26140, Heidelberg, Germany) and monitored continuously with media change every 2-4 days.

Three-Dimension Amnion Epithelial Cells, Culture Spheroid Formation, and Treatment with Cyanidin
To culture the hAECs, we employed the 3D culture Plate system (Elplasia ™ , Kuraray Co. Ltd., Kurashiki, Japan). Spheroids were formed by seeding 1 × 10 6 AECs in Placenta Basal Epithelial Cell Medium into each well of the 24-well plate. The initial culture was maintained for 24 h. After the initial 24 h culture, the medium was changed with 20 µM of Cy3G (Tokiwa Phytochemical Co. Ltd., Chiba, Japan; purity on HPLC ≥ 98%) every 48 h three times for the treatment samples (Day 2, 4, and 6). Control samples were maintained in the Placenta Epithelial Cell Basal Medium that was also changed every 48 h (Day 2, 4, and 6). Finally, we collected RNA samples from the Cy3G-treated and control AECs on day 7.

RNA Extraction and Microarray Analysis
Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan), according to the manufacturer's instructions. The amplified RNA (aRNA) was synthesized using the Gene Chip 3 IVT PLUS Reagent Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Hybridization was achieved using the Affymetrix GeneChip Human Genome U219 Array Strip (HG-U219, Thermo Fisher Scientific). The images were obtained by the GeneAtlas ™ Imaging Station and analyzed using the GeneAtlas ™ Workstation (Thermo Fisher Scientific), according to the manufacturer's instructions. Microarray expression profiling was conducted for two biological replicates of Cy3G-treated samples (Cy7) and untreated control samples (D7).

Ethics Approval
In the present study, we have used hAECs that were preserved at the Tsukuba Human Tissue Biobank Center (THB). THB was established at the University of Tsukuba in November 2013 with an aim to reserve human biospecimens to promote medical research [56,57]. The protocol for isolation, collection, and use of hAECs and other biospecimens was approved by the Ethical Review Committee of the University of Tsukuba Hospital (approval code: H27-58, approval date: 10 July, 2015). Informed written consent was obtained from the mothers who donated the placenta.

Institutional Review Board Statement:
The protocol for isolation, collection, and use of hAECs and other biospecimens for research purposes was carried out in accordance with the Japan's Ministry of Education, Culture, Sports, Science and Technology's "Bioethics and Safety Initiatives" and was compliant with the guidelines of the Declaration of Helsinki. The protocol was approved by the Ethical Review Committee of the University of Tsukuba Hospital (approval code: H27-58, approval date: 10 July 2015).
Informed Consent Statement: Informed written consent was obtained from the mothers who donated the placenta. Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files. Microarray data are deposited in the Gene Expression Omnibus (GEO) under Accession Number: GSE148776 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE148776, accessed on 16 April 2020).